Pneumocystis carinii (PC) pneumonia remains a serious complication of HIV infection and other immunocompromised states including patients undergoing chemotherapy and solid organ transplant recipients. In the setting of HIV infection there is a well-known inverse relationship between CD4+ lymphocyte count and the risk of both PC infection and bacterial pneumonia. In the last funding period we have defined the effector T-cell lineages required for the protection against PCP as well as developed molecular adjuvants using gene products expressed by CD4+ T-cells to investigate whether these molecules could proyide B cell help in the absence of peripheral CD4+ lymphocytes. Using DNA vaccination with an immunodominant antigen identified in the last funding cycle, KEX1, we have screened IFNg (Thi), lL-4 (Th2), IL-17 and IL-21 (Thi 7) as well as CD40L (expressed on activated T-cells) to elicit anti-PC antibody responses, and protection against PCP in CD4-depleted mice. Among these molecules, CD40L was the most effective on eliciting antigen specific IgGi and lgG2a responses in CD4 depleted mice when incorporated in the DNA vaccine platform. We have developed a prime boost vaccination platform that demonstrates efficacy against PC in CD4-depleted mice a codon optimized immunodominant antigen from PC, miniKexin, and CD40L as a molecular adjuvant. Both components were required for vaccine efficacy in the setting of CD4 deficiency. These results lead us to hypothesize that activation of CD40 signaling in vivo, and vaccination with miniKexin can result in antigen specific immune responses and mucosal boosting can provide effective vaccination against PCP in the absence of CD4+ T-cells. Aim 1: If our hypothesis is correct then co-administration of CD40L with mini-Kexin vaccination will result in a CD4 independent humoral response and protection against PC in vivo. Aim 2: Our hypothesis also predicts that this strategy requires endogenous IL-23 and IL-21, and results in durable vaccine responses in both CD4+ T-cell deficient mice and CD40L knockout mice. Aim 3: Test the hypothesis that CD41ND pathogen specific immune responses against P. carinii kexin can be generated in an SIV model of immunodeficiency in macaques.